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Related Concept Videos

Nuclear Export of mRNA02:31

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Before mRNAs are exported to the cytoplasm, it is crucial to check each mRNA for structural and functional integrity. Eukaryotic cells use several different mechanisms, collectively known as mRNA surveillance, to look for irregularities in mRNAs. Irregular or aberrant mRNA are rapidly degraded by various enzymes. If a defective mRNA escapes the surveillance, it would be translated into a protein which would either be non-functional or not function properly. One of the primary irregularities in...
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Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
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An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes
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Strategies for Generating RNA Exosome Complexes from Recombinant Expression Hosts.

Eva-Maria Weick1, John C Zinder1,2, Christopher D Lima3,4

  • 1Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Methods in Molecular Biology (Clifton, N.J.)
|November 27, 2019
PubMed
Summary

This study details methods for purifying and reconstituting eukaryotic RNA exosome complexes from yeast and human cells. These in vitro reconstituted complexes are crucial for understanding RNA processing and degradation pathways.

Keywords:
ExoribonucleaseRNA decayRNA exosome

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The eukaryotic RNA exosome is a vital multiprotein complex involved in RNA processing and degradation.
  • Current understanding relies heavily on genetic and cell biology studies, with in vitro reconstituted complexes offering complementary insights.

Purpose of the Study:

  • To provide a detailed overview of methods for purifying exosome components from recombinant sources.
  • To present protocols for reconstituting functional exosome complexes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Homo sapiens.
  • To discuss the limitations and highlight key discoveries enabled by reconstituted exosome complexes.

Main Methods:

  • Purification of exosome components from recombinant sources.
  • In vitro reconstitution of functional exosome complexes.
  • Detailed protocol development for yeast (S. cerevisiae, S. pombe) and human (H. sapiens) exosome reconstitution.

Main Results:

  • Successful purification of individual exosome components.
  • Reconstitution of active exosome complexes from purified components.
  • Demonstration of the utility of reconstituted complexes in studying RNA metabolism.

Conclusions:

  • Reconstituted RNA exosome complexes are powerful tools for biochemical and mechanistic studies.
  • These methods facilitate a deeper understanding of RNA degradation and processing.
  • The study provides valuable protocols for researchers in the field.